USE OF 2-CHLORO-1-[[4-[(2,6-DICHLOROPHENOXY)METHYL]-PHENYL]METHOXY]-4-METHOXY-BENZENE FOR THE SELECTIVE TREATMENT OF ENTEROVIRAL INFECTIONS IN HUMANS

Abstract: The use of 2-chloro-1-[[4-[(2,6-dichlorophenoxy)methyl]phenyl]methoxy]-4-methoxyb enzene for the preparation of a medicament for the selective therapeutic and prophylactic treatment of enteroviral-caused infections in humans is disclosed.

Description: of 2-cMoro-l-[[4-[(2,6-dichlorop benzene for the selective treatment of enteroviral infectLcms in humans. Introduction to the Invention This invention relates to use of 2-chloro-l-[[4-[(2,6-dichlorophenoxy)methyl]- pheήyl]methoxy]-4-methoxybenzene for the preparation of a medicament for the selective treatment and/or prevention of enteroviral-caused infections in humans. Enteroviruses are members of the RNA-containing Picomavirus Family listed i Table 1 and are known to cause a wide variety of diseases and deaths in humans . drug may exhibit in vitro and in vivo activity against enteroviruses. However, to be clinically useful, such a drug must have broad activity within the enterovirus genus since the same disease may be caused by a variety of different enteroviruses (see Table 2). Although about 70 immunotypes of enteroviruses are known, surveillance data from the Centers for Disease Control (CDC) on nonpolio-enteroviruses from 197 to 1983 show that enteroviruses from the following 15 common immunologic groups account for 65-89% of the isolates for a given year in the United States: echoviruses (echo) 3,4,5,6,7,9,11,24 and 30; coxsackievirus A (CVA) 9; and coxsackieviruses B (CVB) 1 ,2,3,4, and 5. Most recent surveillance data indicate that the following 6 of th original common 15 enteroviral immunotypes represented 64% of all enterovirus isolates in 1988: echo 11 (18.6%), echo 9 (14.1%), CVB4 (10.6%), CVB2 (9.2%), ech 6 (6.2%), and CVB5 (5.1%). As recently as the summer of 1991 , the CDC reported a epidemic in the eastern United States of aseptic meningitis associated with echo 30 which was isolated from patients at a frequency 14 times greater than the normal frequency of isolation. There is no commercially available medicament for treating and/or preventing diseases caused by enterovirus. Thus, there is a need for a medicament for the treatment and even prevention of enteroviral-caused infections in humans. Summary of the Invention This invention provides the use of 2-chloro-l-[[4-[(2,6-dichlorophenoxy)methyl]- phenyl]methoxy]-4-methoxybenzene represented by formula I for the preparation of a medicament for the of treatment and/or prevention of enteroviral infections in humans. I The present invention also provides a pharmaceutical composition for selectively treating and/or preventing enteroviral infections in humans which comprises an anti-enterovirally effective amount of 2-chloro-1-[[4-[(2,6- dichlorophenoxy)methyl]phenyl]methoxy]-4-methoxybenzene represented by formula I suspended in a vegetable oil and optionally containing at least one pharmaceutically acceptable excipient. This invention also provides a method of selectively treating and/or preventing enteroviral infections in humans caused by an enterovirus selected from the group consisting of coxsackieviruses A and B, echoviruses, enteroviruses and polioviruses comprising administering to a human in need of such treating and/or preventing an anti-enterovirally effective amount of 2-chloro-l-[[4-[(2,6-dichlorophenoxy)methyl]- phenyl]methoxy]-4-methoxybenzene represented by formula I or a pharmaceutical composition thereof. This invention further provides a method of selectively treating and/or preventing an enteroviral infection in humans selected from the group consisting of enteroviral respiratory diseases, enteroviral aseptic meningitis, enteroviral meningoencephalitis, chronic enteroviral infections in a gammaglobulinemics and acute hemorrhagic conjunctivitis caused by enterovirus-70 which comprises administering to humans in need of such treating and/or preventing an antienterovirally effective amount of 2-chloro-l-[[4-[(2,6- dichlorophenoxy)methyl]methoxy]-4-methoxybenzene and represented by formula I Detailed Description of the Invention To survey the enteroviral spectrum of the compound of formula I, recent human isolates of enteroviruses representing the above-listed 15 common enteroviral immunotypes were tested for susceptibility using the plaque assay. Older laboratory strains of enteroviruses and less frequently isolated enteroviruses as well as hepatitis A virus also were surveyed. A list of the antiviral activities of the compound of formula against 154 enteroviruses from the 15 common enteroviral immunotypes is presente in Table 3. Again, eighty percent of the 154 enteroviruses (123/154) the compound o formula I exhibited IC5 0 values equal to or less than 0.90 μg ml (2J2 μM) (ECβo = 0.9 μg/MI). While the average IC5 0 value (arithmetic mean) for the compound of formula I against all 154 enteroviral immunotypes was less than 1 μg/ml, the number of the isolates tested in two enteroviral immunotypes (echo 9 and echo 24) was too few (2 isolates) to obtain an accurate representation of the average IC5 0 value. Based on results in a variety of relevant cell culture-based test systems, the compound of formula I is a potent, broad spectrum, orally-active anti-enteroviral agent which has significant activity against enteroviruses believed to be responsible for mos symptomatic enteroviral infections in the United States. Similar activity is expected against enteroviral infections found in other countries. Enteroviruses from the 15 common immunotypes, listed in Table 3, were categorized according to the enteroviral disease diagnosed at the time of isolation. The majority of these isolates (71% or 110/154) were isolated from patients diagnose with asceptic meningitis and/or meningoencephalitis, and the average IC5 0 value of the compound of formula I for this group was 0.98 μg/ml (2.31 μM). The next largest category (18% or 28/155) comprised enterovirus isolated from undiagnosed individuals, and the remaining viral isolates were obtained from patients with enteroviral diseases other than those associated with the central nervous system (CNS). For the latter groups, the average IC50 values for the compound of formula I were based on too few viruses to represent accurate estimations. Ninety-three isolates of the aseptic meningitis/meningoencephalitis were associated exclusively with patients diagnosed as having aseptic meningitis. Of thes 93 isolates, 47% (44/93) comprised isolates from the cerebrospinal fluid, while the remaining isolates were derived from the throat, rectum, stool, or urine. Since the frequency of enterovirus isolation in asymptomatic individuals in this study was 3%, it was concluded that viruses isolated from non-CNS sites in patients with aseptic meningitis likely represented the virus responsible for CNS disease. The antiviral activities of the compound of formula I were determined against laboratory strains of enteroviruses and less frequently isolated enteroviruses as well as hepatitis A virus (HAV), which is classified in its own genus. The average IC5 0 of the compound of formula I against 30 enteroviruses (including polio 1 and polio 2) w 0.97 μg/ml (2.29 μM) and an ECβo 0.8 μg/ml (1.89 μM) was obtained for these viruses Although some of the viruses are less frequently isolated in the United States, severa of these viruses are of considerable importance to other countries and especially those in tropical climates. For example, poliomyelitis in populations not receiving the poliovirus vaccine remains a major health problem. Another example is enterovirus 70, a major cause of acute hemorrhagic conjunctivitis. For both polio 1 , polio 2, and enterovirus (entero) 70, the compound of formula I is inhibitory: five strains of polio 1 (including recent human isolates) had IC5 0 values ranging from 0.009 to 0.04 mg/ml (0.021-0.094 μM); the average IC5 0 for the polio 2 EFI strain in HeLa cells was 0.02 μg/ml (0.047 μM); and 4 strains of entero 70 were inhibited in a range of 0J to 0.4 μg/ml (0.24-0.9 μM). Unlike the situation with the common isolates in which the compound of formula I was inactive against only 2% (3 158 at an IC5 0 > 10 μg/ml) of viruses tested, the compound of formula I was inactive against 19% (7/37) of less frequent isolates and laboratory strains at the maximum concentration tested. This result is largely due to the inactivity of the compound of formula I against isolates of coxsackievirus A. Without testing additional coxsackievirus A imunotypes, it is not possible to establish whether the activity of the compound of formula I against these viruses is representative of other viruses in these immunotypes. Thus, based on the antiviral activities against the 15 common and less common enterovirus isolates as well as activities against laboratory strains of enteroviruses, it is anticipated that the compound of formula I will be active against the majority of enteroviruses encountere in the clinical setting. Either HeLa (human), RD (human rhabdsarcoma), or BGMK (buffalo green monkey) cells were used to determine the antiviral activities of the compound of formula I against all enteroviruses which were tested. Thus, a therapeutic index ("Tl's'Jfor the compound of formula I with each virus was calculated by measuring adverse effects of the compound of formula I in these cell lines using the CPE-based MTT assay. The term "therapeutic index" as used herein is defined as cytotoxicity IC value divided by the antiviral IC5 0 value. Of 184 enteroviruses exhibiting measurabl activity in the plaque assay, only 8.7% had Tls <10 and 5.4% had Tls > 11 and < 25, while 75J% has Tls > 51. Therefore, for the majority of enteroviruses tested, the antiviral activity of the compound of formula I is independent of possible adverse effects of the compound of formula I on cells and cytotoxicity does not contribute to th antiviral activity of the compound of the formula I. The compound of formula I is efficacious against both echovirus-4 ("echo 4") and coxsackievirus A9 ("CVA9") viral infections when orally administered to mice in a therapeutic (+3.5 hr.) regimen at 20 mg/kg/day (4 doses of 5 mg/kg). The compound formula I orally administered in this therapeutic regimen to mice intracranially infecte with echo 4 at 980 PFU, exhibited an area under the curve (AUC) activity at day 21 of 75.2%. Mice treated with the compound of the formula I in a therapeutic regimen infected intracranially with CVA9 at 1,200 PFU have an AUC activity of 71.7%. In ech 4 infected mice, the AUC activity decreases from 75.2% to 34.4% as the infectious challenge increases 3 orders of magnitude from 980 PFU to 980,000 PFU. Similarly, in CVA9 infected mice, the AUC activity decreases from 71.7% to 35.8% as the infection challenge increase 3 orders of magnitude from 1,200 PFU to 1,200,000 PFU The compound of formula I was also effacious against the polio-2-viral infectio in the murine model in a therapeutic regimen when oral administration of the compound of formula I is 3.5, 6, and 24 hours after the mice were infected intracranially with polio-2 virus. Prophylactic treatment of mice by the oral administration of the compound of formula I, 24 hours before infection with echo-4, CVA9 and poliovirus-2 produced a similar effacious result compared to those in the three therapeutic regimens describe above. In two prophylactic treatment experiments (-24 hr) with 2 oral doses of 30 mg/kg (60 mpk/day) of the compound of formula I, mice infected with 98,000 PFU of Echo-4 and 1,200,000 PFU of CVA9 exhibited AUC activity of 59.0% and 50%, respectively. Prophylactic treatment with the compound of formula I, begun 24 hours (-24 h before intracranial infection into mice of 520,000 PFU of poliovirus-2, provided simil efficacy when the compound was orally administered BID (2 x 20 mg/kg (40 mpk/da AUC = 46.4%), TID (3 x 6.7 mg/kg (20 mpl/day), AUC = 29.2%) and QID (4 x 5.0 mg/ (20 pmk/day), AUC = 37.2%). Serum levels after oral administration of the compound of formula I in corn oil dosages of 1 , 5, 20 and 30 mg/kg were measured in mice, rats, dogs, and monkeys. All species showed a dose-related increase in AUC, although the AUC did not increase at the higher dose. Similarly, the CM AX increases with the dose of the compound of formula I in all species. Among the four species, the highest serum levels of the compound of formula I occurred in beagles. The serum half-life of the compound of formula I was 4.7 hours in mice, but was dramatically greater in the higher species: 26 hours and > 24 hours in beagles and monkeys, respectively. Th time to CMAX (i.e., TMAX) had a similar value in mice, rates and dogs (i.e., 2-4 hrs.), but it was longer in monkeys (8-12 hrs.). The shorter TMAX in dogs suggests that th serum life of 26 hrs. is independent of absorption in this species. On the basis of the pharmacokinetic results in dogs, the expected human levels of the compound of formula I should achieve antiviral levels (1 mg/ml) for 24 hours at a single dose of 10 mg/kg administered orally to humans. A two week oral pilot toxicity study was conducted with the compound of form I in rats and dogs. The LD50 value was in excess of 1000 mg/Kg day in the rat and was in excess of 750 mg/Kg/day in the dog after two weeks of oral dosing. Materials and Methods Enteroviruses, representing the 15 most commonly isolated immunotypes in the USA were obtained from Centers for Disease Control (CDC), Atlanta, Georgia. Normally, five viruses from each immunotype were obtained. Other, less frequently isolated enteroviruses and the rhinoviruses were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland. Enteroviruses and rhinoviruses were propagated in an appropriate cell line, eg HeLa, (human), BGMK (Buffalo greemh monkey), or RD (human rhabdosarcoma) cells and working stocks were prepared as cellular lysates. The standard procedures used for quantitating antiviral activity of the compoundd of formula I in vitro and in vivo are given herein below. Pre-mix plaque assay: 150 plaque forming units (PFU; infectious particles) were mixed with test compound (0.0001-50 mg/ml) in Eagles' modified minimal essential medium (EMEM) for 45 min. and added to monolayers of appropriate cells. After 45 min. at 33°C, the inoculum was aspirated, the cells were washed, and plaque analysis was performed. In accordance with the methods desclosed by Trousdale, M.D., et al., (1977) in Biochem. Biophys. Res. Commun. 76:368-375 and Rueckert, R.R., et al., (1981) in Methods in Enzymology 78:315-325. The number PFU was determined at each concentration and plotted as a percentage of control PFU using Cricket Graph ® or Wingz ® graphics programs and the individual values were connected by a line through each value. To determine the IC5 0 concentration, a perpendicular line from the intersection of the PFU curve and a horizontal line representing 50% inhibition were connected to the abscissa. The value on the abscissa which intersects the perpendicular line represents the IC50 value. Values indicative of percentage inhibition (ordinate) are represented linearly, while the concentrations of test molecule (abscissa) are represented logarithmically. Pre-mix cytopathic effect (CPE) assay: This assay, based on viral cytopathology, has a colorimetric endpoint dependent on light absorption by MTT(3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SigmaChemical Co.) formazan. Briefly, test compound and virus were premixed for 45 min. at 22°C and used to infect cells {multiply of infection (MOI) = 1.0} in 96 well microelisa plates. Following 45 min. incubation (37°C), the culture fluid is aspired and the monolayers washed once with phospate buffered saline (PBS), EMEM with 1% fetal calf serum (FCS) is added, and incubation is continued for 18-24 hr. prior to measuring cellu® r graphics programs as described for the pre-mix plaque assay. CPE cytotoxicity assay using MTT: The 50% inhibitory concentration (IC50), referred to as the cytotoxic IC 50 was determined as follows: Uninfected monolayers of cells in 96-well microelisa plates were treated with the compound of formula I (0.025-50 mg/ml) for prescribed periods of time prior to lysis of the monolaye and treatment with MTT. The IC50 value is determined as described for the pre-mix plaque assay Therapeutic index: The therapeutic index "Tl" of the compound of formula I, which is dependent on the enterovirus tested as well as the type of cell in which the test was performed, was determined by two procedures: (i) The antiviral IC5 0 was determined by pre-mix plaque assay and the cytotoxic IC50 was determined by the MTT assay. In the latter, test molecule was present for 45 min. prior to removal by aspiration and the period of incubation prior to MTT treatment was identical with that o cells used in the plaque assay, (ii) The antiviral IC 50 and the cytotoxic IC50 values were determined by CPE assay using MTT to determine the endpoints. Since enteroviruses normally adversely effect cellular metabolic processes to result in a cytopathic effect during their replication, the MTT assay is suitable for quantitating the antiviral effect of a test agent by indirectly assessing cellular metabolic functions. Thus, at concentrations in which an antiviral agent inhibits viral replication, relatively more cells remain viable as measured by the MTT assay compared to a less effective antiviral agent that permits comparatively more viral replication and subsequent killin of cells. The MTT assay is particularly advantageous for determination of Tl since the virus and the antiviral molecule are tested under identical conditions with an equivalent end-point methodology. Activity in Animals: Studies with mice (as well as other animals) were performed as recomended in the Guide for the Care and Use of Laboratory Animals (NIH Publications 85-23). Groups of 15-25 male mice (16-20 g; Harland Sprague Dawley) were infected intracranially with poliovirus 2, echovirus 4, or coxsackievirus A9 as described in McKinlay and Steinberg (1986 Antimicrob. Agents Chemothen. 29: 30-32). Treatment with the compound of formula I was by oral gavage (0.3 ml in corn oil) or other oleogacious materials such as peanut oil could also be used. Survival was monitered daily for 21 days. Results were evaluated for statistical significance using Chi square analysis, and protection was considered to be significant if results met a probability of p < 0.05 for at least 5 days during the study compared to the placebo-treated group. An additional means of efficacy called area under the curve activity ("AUC activity") could also be us«d. AVC activity is calculated from the formula: AUC activity = [(experimental AUC-placebo AUC) divided by (maximum AUC-placebo AUC) x 100]. Dosing Formulation: in a preferred aspect of the present invention, a 2 mg/ml stock of the compound of formula I was prepared as a suspension in com oil for studies in which the compound was administered orally. On each day of the study, the stock was diluted with the correct amount of corn oil to give the appropriate mg/kg dosage based on the average weight of the mice for that day. In another preferred aspect of the present invention, the amount of the compound of formula I which may be suspended in one milliliter of com or peanut oil to form a pharmaceutical compositions is in the range of about 100mg to about 1 gram The pharmaceutical compositions of the present invention may also contain the typical pharmaceutically acceptable excipients, e.g., stabilizers, surfactants and antioxidants. Typical antioxidants include butylated hydroxyanisole "BHA" and butylated hydroxytoluene "BHT" which are used to prevent vegetable oils from becoming rancid. Stabilizers, e.g., lecithin and surfactants, e.g., polysorbate 60 or Arlacel 186 (a mixture of glyceryl oleate and propylene glycol available from ICI Americas, Inc., Wilimington, Delaware) may also be added. In other studies where methycellulose (0.4% methylcellulose, 0.9% benzyl alcohol, 0.9% NaCI & 0.5% polysorbate-80), 1% gum tragacanth, or 1% lecithin were used as the oral dosing vehicle, the compound of formula I was added as a powder to the vehicle, mixed by homogenization (one min) and the resulting suspension orally administered to the animals. Food matrix was prepared by mixing the compound of formula I in 1% lechitin and then adding it to a hot solution of 2x Jello ® cherry flavored gelatin (but any gelatin could be used) plus sifted ground food (Purina® Mouse chow). The final concentratio of the compound of formula I in the matrix for a 10 mg/kg dose was calculated based on a retriction of 10 grams of food a day per mouse (a 1 mg/kg dosage was present in each g of food matrix). In the efficacy studies, food matrix was made every four days and corrected for the weight change in the groups. Food was placed in trays at the bottom of cages for consumption. The compound of formula I was prepared by the below-described Williamson ether synthesis as described in Example 15a of Orally Active Antiviral Compounds, International Publication No. WO 92/22520, published 23 Dec. 1992, International Appln. No. PCT/US92/04961. A. Add 5g of 2-chloro-4-methoxyphenol to a mixture of 17g of α,α'-dibrom p-xylene to a stirred mixture of 2.5g of 50:50 (w/w) NaOH:H2θ and 50mL of DMF. Sti the so-formed mixture for 4 hours. Partition the reaction mixture with methylene chloride and water. Separate the organic layer and remove the solvent by rotary evaporation to obtain a solid residue. Purify the residue on a silica gel chromatography column using pure hexane to pure methylene chloride as the eluant to provide 8g of 1-[4-bromomethylbenzyloxy]-2-chloro-4-methoxyphenyl ether. B. To a solution of 0.50g of 2,6-dicholorophenol in a mixture of 0.12g of NaOH in 5m L of DMF, add 0.50g of the phenyl ether from Step (A) and stir for 4 hour at room temperature. Add 50mL of water to the reaction mixture and filter the crude precipitate so-formed. Purify the crude precipitate by crystallization from methylene chloride to provide 350 mg of 2-chloro-1-[[4-[(2,6-dichlorophenoxy)methyl]- phenyl]methoxy]-4-methoxybenzene, the compound of formula I. as a pure solid. The compounds of formula I may be administered by any conventional mode o administration by employing an antienterovirally effective amount of a compound of formula I for such mode. The dosages may be varied depending upon the requirements of the patient in the judgement of the attending clinician, the severity of the condition being treated and the particular mode of administration being employe Determination of the proper dosage for a particular situation is within the skill of the a Treatment can be initiated with similar dosages which are less than the optimum dos of the compound. Thereafter, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. The compound of formula I may be administered for example, orally, intravenously, intraventricularly, subcutaneously, intramuscularly, or via rectal suppository; intravenous dosage should preferably comprise continuous drip infusion The pharmaceutical composition of this invention contains an anti-enterovirall effective amount of the compound of formula I suspended in a vegetable oil, e.g., cor oil or peanut oil and optionally a pharmaceutically aceptable excipient. The preparation of pharmaceutical compositions and the selction of pharmaceutically acceptable excipients are well known to those skilled in the art; see for example Remington's Pharmaceutical Sciences, Mark Publishing Co., Easton, Pa., 15th editio (1975) or International Publication WO 92/2250, published 23 Dec. 1992. The preferred carrier is corn oil which may be used alone or in combination with stabilizer e.g., lecithin, or surfactants, e.g., polysorbate 60 or Arlacel 186. The anti-enterovirally effective amount of the compound of formula I is in the range of about 1 to 90 mg/kg of body weight per day. A dose of about 10 to 15 mg/kg given one to six times a day, in equal or divided doses is preferred. A dose of 10 mg/kg day is more preferred. The exact dosage of the compounds of this invention which is administered is dependent, in the judgement of the attending clinician, upon a variety of factors, e.g. the age and weight of the individual being treated, the mode of administration, the potency of the administered compound, the indication for which the durg is administered and the severity of the ailment being treated. Because of the broad anti-enteroviral activity of the compound of formula I, the high degree of potency of the molecule against enteroviruses, the efficacy afforded to enterovirus-infected mice following oral administration of the molecule, and measurable blood levels in dogs and primates following oral dosage of the molecule, we propose that the compound of formula I will have utility in treating and/or preventing human enterovirus infections, including the diseases listed in Table 2. In a preferred aspect of this invention, the effective therapeutic and/or prophylactic treatment of the following enteroviral diseases in humans by the compound of formula i or a pharmaceutical composition thereof is expected: enteroviral respiratory disease (ie., summer cold, summer grippe), enteroviral aseptic meningitis, enteroviral meningoencephalitis, chronic enterovirus infection in agammaglobulinemics, or acute hemorrhagic conjunctivitis caused by enterovirus-70. Patient Population: Selection of a patient population experiencing enterovirus disease would be based on: (i) pathognomic symptomatology associated with some enterovirus diseases (e.g., acute rhinitis/cold symptoms during the summer, skin lesions in hand-foot-and-mouth diseases, vesicle in pharynx, or myalgia associated with pleurodynia); (ii) confirmation of enterovirus infection by isolation of virus using cell culture techniques ; (iii) PCR (polymerase chain reaction) diagnosis of viral genetic material (e.g., in cerebral spinal fluid for enteroviral aseptic meningitis); immunological techniques such as fluorescent antibody enzyme-linked imunoabsorbant technolgy; or a combination of the above. Efficacy Evaluation: Evaluation of the efficacy of the compound of formula I in humans affected with enteroviral disease is measured by reduction of symptomatology and/or the viral tissue load associated with a particular enteroviral illness. For example, parameters that could be measured to assess efficacy include: (i) decrease of symptoms associated with summer cold; decrease in shed virus, decrease in mucous weight, decrease in fever, and decrease in myalgia compared t placebo-treated individuals; (ii) for aseptic meningitis: reduction of time in hospital, reduction of headache & fev«r, decrease in time to normal neurological findings compared to placebo patients; (iii) for hemorrhagic conjunctivitis: improvement in sight, clearing of conjunctiva more rapidly compared to placebos. Other parameters t assess the efficacy of the compound of formula I may need to be established for each enteroviral disease under clinical testing, in the opinion of the attending clinician. Summary of Results Against 154 enteroviruses representing 15 common isolated enteroviral immunotypes, the compound of formula I exhibits an average IC5 0 (50% inhibitory concentration) of 0.98 μg/ml (2.31 μM) in the plaque assay (Table 3). The average IC5 0 for 30 less frequently isolated enteroviruses and laboratory strains of enteroviruses, including poliovirus (polio) types 1 and 2, is 0.97 μg/ml (2.29 μM). Using the MTT assay as a measure of adverse effects produced by the compound of formula I in uninfected cells, no adverse effects were observed in cell cultures at concentrations of the compound of formula I equal to or greater than 51 times the antiviral IC5 0 concentrations for 75% of the enteroviruses tested (138 of 184 viruses, including common clinical isolates and laboratory strains of enteroviruses). Thus, the antiviral activity of the compound of formula I is not due to cytotoxic effects of the compound. Against rhinoviruses, which represent another genus of picomaviruses, the compound of formula I exhibited an activity (IC 50 ) equal to or less than 10 μg/ml (23.6 μM) agains 38% of rhinoviruses which were surveyed. Representing a third genus of picomaviruses, a single strain of hepatitis A virus was inhibited by the compound of formula I at an IC50 value of 10.1 μg ml (23.8 μM). The compound of formula I was inactive against other RNA viruses or DNA viruses comprising ten viral families which were tested, including influenza virus, human immunodeficiency virus, herpes simplex virus, and adenovirus. The compound of formula I was orally active prophylactically (24 hrs. before infection) and therapeutically (3.5 hrs. after infection) against Echovirus 4, Coxsackievirus A9, and poliovirus 2 infections in murine models. The compound of formula I also exhibited increased activity in vivo as the size of the viral inoculum decreased and it reduced viral tissue titers in mice in both therapeutic and proprophylactic regimens. The compound of formula I is orally bioavailable after a single oral dose: In mice receiving 1 mg/kg, oral absorption was 78% and absolute oral bioavailability wa 50%. After oral administration to mice, the compound of formula I reaches inhibitory levels in the brain, the primary site of viral infection in the murine models. Blood level of the compound of formula I are also achieved in mice, rats, dogs, and monkeys following oral dosage. The half-life of the molecule in the serum increases from 4.7 hours in mice, to 26 hours in dogs. Based on the antiviral activity of the compound of formula I in cell culture and in animal models and the ability to achieve significant blood and tissue levels in mice, rats, beagles, and cynomolgus monkeys following oral administration of the molecule, efficacious clinical behavior in humans for the therapeutic and prophylactic treatment of enteroviral infections in humans is expected. Table 1. Taxonomy of the the Picornavirus Family. Group or Viral No. ofa Memberb Genus Immunotypes Subgroup Viruses Viruses Enterovirus 0 coxsackieviruses A 23 CVA1-22, 24 B 6 CVB1-6 echoviruses ..d 30 Echo 1-9. 11-27, 29-34 enteroviruses -d 4 Entero 68-71 polioviruses „d 3 Polio 1-3 Vilyuisk virus e ~d 1 Vilyuisk Rhinovirus human rhinovirus A 33 _f B 67 ..f Hepatitis A hepatitis A virus ..d 1 Hepatitis A Apthovirus foot-and-mouth-9 -d 7 FMDVA.CO. disease virus SAT 1,3.3, Asia-1 Cardiovirus encephalomyo- n EMC 6 EMC. MM carditis (EMC) virus mengo, ME, SK, Columbia Theiler's murine-' TMEV 7 GDVII, FA DA, encephalo- WW, T04, Yale. mvelitis virus (TMEV) BeAn 8366 Legend to Table 1. a Number refer to the number of viruses or number of viral immunotypes in a subgroup. D Abbreviations for the names of viruses are shown. In instances where numbers or viruses are missing (e.g.. CVA23), viruses having these numbers originally were incorrectly classified in the enterovirus genus. Upon reclassification, the numbers were dropped from the classification system foe enteroviruses (Rueckert, 1990). c Enteroviruses which infect non-human species as their natural host are not included in this listing. d Viral subgroups do not exist. e Vilyuisk virus is associated with a neurodegenerative disease occurring in humans in Siberia (Casals, 1963; Lipton et al., 1983). Legend to Table 1. (continued) f Although 100 immunotypes of human rhinoviruses are officially recognized by the International Committee on the Taxonomy of Viruses, at least 10 more rhinoviruses have been reported. Classification of individual rhinoviruses into groups A and B can be found in Andries et al. (1992). 9 Foot-and-mouth-disease viruses do not infect humans. n EMC viruses have rarely been isolated from humans, however they are not associated with any apparent disease (Lipton & Rozhon, 1986). ' Theliler's murine encephalitis viruses infect only mice (Lipton & Rozhon, 1986). Table 2. Clinical Spectrum of Enteroviral Disease 8 Principal Disease Viral Immunotypes Diseases of Proven Enterovirus Etiology respiratory disease (summer colds; many coxsackievirus A & B and echovirus summer grippe) immunotypes pneumonia, pneumonitis, CVA 9 & 16, CVB 4 & 5; echo 68 bronchiolitis aseptic meningitis CVA 1-11, 14, 16-18, 22, 24; CVB 1-6; all echo except types 24, 26, 29, 32; polio 1-3 meningoencephalitis CVB 1-5; enterovirus types 68-72 encephalitis CVA 2, 5, 6, 7, 9; CVB 1-3, 5, 6; echo 2, 6, 9, 19, others less frequently chronic infection in agamma- echo 2, 3, 5, 9, 11 , 19, 24, 25, 30, 33 globulinemics poliomyelitis, paralysis, paralytic myelitis polio 1-3; CVA 4, 6, 7, 9, 11, 14, 21; CVB 1-6; echo 1-4, 6, 7, 9, 11, 16, 18, 19. 30; entero 70, 71 myocarditis, pericarditis, dilated CVB 1-5 cardiomyopathy exanthems (rubelliform, roseolifom, CVA 2, 4, 5, 9, 16; CVB 1 , 3, 4, 5; echo 9, herpetiform) 16; other less frequent echo types sepsis, systemic infection in neonates & CVB 1-5; echo 6, 9, 11, 14, 19, 31; CVA 2, infants 9, 16 less frequently Guillian-Barre syndrome echo 2, 6, 9, 19; possible other echo types hepatitis, pancreatitis CVA 4, 9; CVB 5; echo 4 & 9; also same viruses that cause systemic infection in infants herpangina CVA 2-6, 8, 10, 22 hemorrhagic conjunctivitis enterovirus-70; CVA 24 variant epidemic pleurodynia, myalgia, CVB 1-5; CVA 4, 6, 10 sporadically Bomholm disease Table 2. (continued). Clinical Spectrum of Enteroviral Disease 8 hand, foot & mouth disease CVA 5, 7, 9, 10, 16; enterovirus 71 lymphonodular pharyngitis CVA 10 epidemic myalagia echo 1, 6, 9 Disease of Possible Enterovirus Etiology neonatal diarrhea, general diarrhea CVA 18, 20-22, 24; many echo types postviral fatigue syndrome CVB immunotypes adult onset (insulin-dependent) CVB immunotypes diabetes mellitus a.Abelman, W. H. 1988. The etiology, pathogenesis and physiology of dilated cardiomyopathies. In: New concepts in Viral Heart Disease (H.P. Schultheiss, ed.), pp. 3-21. Springer-Verlag, Berlin, Germany. Grist, N.R., and Reid, D. 1988. General pathogenicity and epidemiology. In: Coxsackieviruses - A General Update (M. Bendinelli and HJ. Friedman, eds.), pp. 221-239.. Plenum Press, New York, New York. Kaplan, M.H. 1988. Coxsackievirus infection in children under three months of age. In Coxsackieviruses - A General Update (M. Bendineli and HJ. Friedman, eds.), pp.241-251. Plenum Press, New York, New York. Kew, O.M., Nottay, B.K., Htch, M.H., Hierholzer, J.C., and Obijeski, JF. 1983. Oligonucleotide fingerprint analysis of enterovirus 70 isolates from the 1980 to 1981 pandemic of acute hemorrhagic conjunctivitis: evidence for a close genetic relationship among Asian and American strains. Infect. Immun. 41: 631-635. Melnick, J.L. 1990. Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In Virology (B.N. Fields and D.M. Knipe, eds.), pp. 549-605. Raven Press Lt ., New York, New York. Modlin, J.F. 1990. Coxsackieviruses, echoviruses and newer enteroviruses. In: Principles and Practice of Infectious Disease (G.L Mandell, R.G. Douglas, Jr., and J.E. Bennet, eds.), pp. 1367-1383. Churchill Livingstone, Inc. New York, New York. Sattar, S.A., Dimock, K.D., Ansari, S.A., and Springthorpe, V.S. 1988. Spread of acute hemorrhagic conjunctivitis due to enterovirus-70. J. Med. Virol 25: 289- 296. Wagenknecht, L.E., Rosemon, J.M., and Herman, W. H. 1991. Increased incidenc of insulin-dependent diabetes-mellitus following an epidemic of coxsackievirus B5. Am. J. Epidemiology 133: 1024-1031. Wright, P. W., Strauss, G. H., and Langford, M.P. 1992. Acute hemorrhagic conjunctivitis. American Family Physician 45: 173-178. Viruses shown in this column represent the most common enteroviruses isolated from individuals diagnosed with each disease, however other enteroviruses, which are not listed as being associated with the disease, may be capable of producing the same disease. Table 3. Anti-Viral Activity of the Compound of Formula I Against 15 Common Clinical Enterovirus Isolates 1 Entereoviral Immunotype 1 Mean 2 IC50 (μg/mL) E3 0.40 E4 0.15 E5 0.93 E6 0.30 E7 0.16 E9 1.45 E11 0.08 E24 0.15 E30 0.42 CA9 0.73 CB1 3.02 CB2 0.31 CB3 0.58 CB4 0.19 CB5 2.80 1. E =Echorirus Immunotype. CA =Coxsackievirus A Immunotype. CB =Coxsackievirus B Immunotype. 2. Antiviral Activity was determined by per-mix plaque assay (see Materials and Methods Section). Table 4. Summary of Cell Culture-Based Activies of the Compound of Formula I Against 15 Common Enteroviruses and Laboratory Strains of Enteroviruses. No. of Enterovirus Viruses No. with IC50 No. with IC50 Average 8 Group Tested < 1 μg/ml ≤ 10 μg ml IC50 EC 8 o b 15 Common 158 125 155 0.98 μg/ml 0.9 μg/ml Isolates 0 (2.31 μM) (2.12 μM) Lab Strains/ 37 24 30 0.97μg/ml 0.8 μg/ml Infrequent (2.29 μM) (1.89 μM) lsolates d a Average IC50 value was calculated from the antiviral activities (pre-mix plaque assay) of 128 viruses from the 15 common enteroviruses isolate group and 30 viruses from the lab strain group. ECβo represents the concentration of the compund formula I that inhibits 80% of the viruses tested based on their IC50 values. c The 15 common enteroviral isolates are listed in Table 3. d Includes polioviruses 1-3 and enterovirus 70.

Claims: What is Claimed: I. The use of 2-chloro-l-[[4-[(2,6-dichlorophenoxy)methyl]- phenyl]methoxy]-4-methoxybenzene represented by formula I for the preparation of a medicament for treating and/or preventing enteroviral infections in humans. 2. The use of claim 1 wherein enteroviral infections in humans caused by an enterovirus is selected from the group consisting of coxsackieviruses A and B, echoviruses, enteroviruses and polioviruses. 3. A method of selectively treating and/or preventing an enteroviral infection in humans selected from the group consisting of enteroviral respiratory diseases, enteroviral aseptic meningitis, enteroviral meningoencephalitis, chronic enteroviral infections in a gammaglobulinemics and acute hemorrhagic conjunctivitis caused by enterovirus-70 which comprises administering to humans in need of such treating and/or preventing an antiehterovirally effective amount of 2-chloro-l-[[4-[(2,6- dichlorophenoxy)methyl]methoxy]-4-methoxybenzene and represented by formula I or a pharmaceutical composition thereof. 4. A pharmaceutical composition for selectively treating and/or preventing enteroviral infections in humans which comprises an anti-enterovirally effective amount of 2-chloro-]-[[4-2,6-dichlorophenoxy)methyl]phenyl]methoxy]-4- methoxybenzene suspended in vegetable oil and optionally a pharmaceutically acceptable excipient thereof. 5. The pharmaceutical composition of claim 4 which comprises 0-10% lecithin. 6. The pharmaceutical composition of claim 4 which is in the form of suspension in com oil or peanut oil for oral administration. 7. The pharmaceutical composition of claim 4 which is contained in a capsule. 8. The pharmaceutical composition of claim 4 which further comprises a pharmaceutically acceptable stabilizer and/or non-ionic surfactant. 9. The use, method or pharmaceutical composition of any preceding claim wherein the compound of formula I is administered orally. 10. The use, method or pharmaceutical composition of any preceding claim wherein the entienterovirally effective amount of the compound of formula I is in the range of about I to about 60mg/kg of body weight per day.

Inventors: BUONTEMPO PETER, COX STUART A, DEMARTINO JASON L, O'CONNELL JOHN F, ROZHON EDWARD J, WRIGHT JACQUELYN M, BUONTEMPO, PETER, COX, STUART, A., DEMARTINO, JASON, L., O'CONNELL, JOHN, F., ROZHON, EDWARD, J., WRIGHT, JACQUELYN, M.
Applicants: BUONTEMPO PETER, CONNELL JOHN F O, COX STUART A, DEMARTINO JASON L, ROZHON EDWARD J, SCHERING CORP, WRIGHT JACQUELYN M, BUONTEMPO, PETER, COX, STUART, A., DEMARTINO, JASON, L., O'CONNELL, JOHN, F., ROZHON, EDWARD, J., SCHERING CORPORATION, WRIGHT, JACQUELYN, M.
Application year: 1994
Main classification: A61K31
IPC codes: A61K9, A61K31, A61K47, A61P31